Improved Apparatus for Compound Separation

The present invention relates to separation of biomolecules from compositions. In particular, the present invention relates to an apparatus for collective sorption and filtration of protein.

Tangential flow filtration is a rapid and efficient method for separation and purification of biomolecules. Tangential flow filtration is also known as crossflow filtration. Tangential flow filtration is characterised by a majority of the feed flow traveling tangentially across the surface of the filter. The main driving force of cross-flow filtration is transmembrane pressure, which is a measure of the pressure difference between the two sides of the membrane. During the feed being passed across the filter membrane a proportion of the material being smaller than the membrane pore size will pass through the membrane as permeate, while the remaining material on the feed side of the membrane is considered as retentate.

During the separation process of biomolecules by tangential flow filtration, the biomolecules may also be bound by adsorbents in order to retain them in the retentate. State of the art is to allow the adsorbent to suspend and recirculate in the retentate and keep the tangential flow filtration membrane free from fouling and build-up of particle layers by applying a high tangential flow and frequent back flushes. However, this may leave varying amounts of target protein/target compound unbound in the composition, which may pass through to the permeate as a loss of product. A fraction of the target compounds are thus frequently wasted during the separation process. This is not optimal from a cost and resource perspective. Also, due to the suspended and recycled state of the adsorbent, considerable amounts of liquids e.g. for the washing and elution process is used in the apparatuses and methods used today for separation. In times of sparse resources, an optimisation of this part of the process would be advantageous.

Hence, an improved apparatus for separation and purification of biomolecules such as proteins would be advantageous, and in particular a more efficient and/or economic process for separation and purification of biomolecules such as proteins would be advantageous.

Thus, an object of the present invention relates to the provision of an apparatus and a process, which optimises the amount of biomolecules, such as proteins, obtained from the separation as well as saves resources during the separation process.

In particular, it is an object of the present invention to provide an apparatus and process that solves the above mentioned problems using tangential fluid flow filtration for separation and purification.

Thus, one aspect of the invention relates to an apparatus comprising a first inlet; a tangential flow filtration unit comprising a membrane having a retentate site and a permeate site and a nominal pore size in the range of 1 to 1000 μm; one or more first fluid connections leading fluid from said first inlet to said tangential flow filtration unit; one or more second fluid connections leading retentate from said tangential flow filtration unit to said first inlet and/or said first fluid connections; a circulation pump for re-circulation of fluid through said tangential flow filtration unit via said first and second fluid connections; one or more third fluid connections for leading permeate to a first outlet; and a sorption material, wherein said sorption material comprising chemically modified porous particles having an average particle size of at least 5 micron, such as at least 10 micron, such as at least 20 micron; and said apparatus, when in use, comprises a dynamic layer of said sorption material on the retentate side of said membrane, said dynamic sorption material layer being capable of reaching a thickness of at least 1 mm, such as at least 3 mm, such as at least 5 mm, such as at least 10 mm, such as at least 15 mm, such as at least 20 mm, such as at least 25 mm, such as at least 30 mm, such as at least 35 mm, such as at least 40 mm.

Another aspect of the present invention relates to an apparatus comprising a first inlet; a tangential flow filtration unit comprising a membrane having a retentate site and a permeate site and a nominal pore size in the range of 1 to 1000 μm; one or more first fluid connections leading fluid from said first inlet to said tangential flow filtration unit; one or more second fluid connections leading retentate from said tangential flow filtration unit to said first inlet and/or first fluid connections; a circulation pump for re-circulation of fluid through said tangential flow filtration unit via said first and second fluid connections; one or more third fluid connections for leading permeate to a first outlet; wherein said apparatus further comprises a permeate pump capable of creating a partial vacuum on the permeate site of the membrane and a valve for inlet or outlet of air to the permeate site of the membrane; whereby the tangential flow filtration unit may be substantially drained or substantially filled with liquid on the permeate site of the membrane by selective controlling of the valve, the permeate pump and the circulation pump; and the tangential flow filtration unit is adapted for acquiring, when in use, a dynamic layer of a sorption material with a thickness of at least 1 mm, such as at least 3 mm, such as at least 5 mm, such as at least 10 mm, such as at least 15 mm, such as at least 20 mm, such as at least 25 mm, such as at least 30 mm, such as at least 35 mm, such as at least 40 mm on the retentate site of the membrane.

Yet another aspect of the present invention is to provide a process for separation of a target compound comprised in a biological composition, the process comprising the steps of:

The present invention will now be described in more detail in the following.

Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

Sorption material

In the present context, the term “sorption material” refers to a material that is able to attach to a specific compound or group of compounds, such as, but not limited to proteins. A sorption material may also be named an adsorbent.

In a preferred embodiment, the sorption material is a protein sorption material.

In the present context, the term “adsorbent” refers to a material, which is able to interact with a specific compound or group of compounds which are thereby reversibly attached to the surface of the material. For example, the adsorbent may comprise ligands able to interact with the specific compound, such as but not limited to: ion exchange ligands, hydrophobic ligands, metal chelating ligands, affinity sorption ligands, and mixed mode ligands. An adsorbent may be substantially impermeable whereby only the outer surface is available for interaction with compounds, or it may be highly porous with pores large enough to allow compounds to diffuse into and interact with the inner surface of the adsorbent.

Alternatively, the adsorbent may comprise an immobilized enzyme which is bound by reversible adsorption, physical entrapment or by covalent chemical bonding to the sorption material and for enzymatic modification of the compound present in the composition, such as but not limited to controlled hydrolysis of proteins in the composition into peptides by an immobilized protease, or modification of carbohydrates e.g. polysaccharides.

In the present context, the term “chemically modified porous particles” refers to natural or synthetic porous particles being treated with one or more chemical derivatisation steps to provide porous particles with desired chemical and/or physical properties e.g. as described herein. The term also includes porous particles produced themselves by chemical reactions leading directly to porous particles having the desired chemical and/or physical properties.

In the present context, the term “dynamic layer of sorption material” refers to a layer of sorption material on the retentate site of the membrane. This layer is dynamic as the thickness of the layer may change over time i.e. during the event of running the separation process.

The thickness of the dynamic layer may vary over the span of the retentate site of the filter. Thus, the thickness of the dynamic layer is to be understood as the average thickness reached by at least a part of the dynamic layer, such as the average thickness reached over the entire retentate site surface of the filter.

In the present context, the term “elution liquid” refers to a liquid substance or composition, which is capable of releasing a compound attached to a sorption material.

In the present context, the term “circulating flow” refers to the flow of the part of the fluid that enters the tangential flow filtration unit and leaves it as retentate that is circulated back to the tangential flow filtration unit, such as through the mixing tank.

In the present context, the term “tangential flow filtration unit” or cross-flow filtration unit, refer to filtration equipment wherein the filter surface is tangential to the flow direction of the composition, which is to be filtered.

In the present context, the term “membrane” refers to a filter or a membrane useful for separating components of a composition.

Membrane and filter are used interchangeably herein.

In the present context, the term “flux” or “flux rate” means the volume of liquid passing one square meter of filter area per hour. The unit applied in the art is LMH (i.e. L/m/h).

In the present context, the term “nominal pore size” relates to the pore size of a filter at which a minimum percentage of components larger than that pore size should be retained by the filter. For example, a nominal pore size of 60% for a filter having an average pore size of X, is considered to retain at least 60% of particles which are larger than X.

In the present context, the term “retentate” relates to a composition which has passed over a filter without passing through the pores of the filter to the permeate side. Thus, a retentate is the part of a filtered composition which was retained by the filter i.e. retained on the retentate side of the filter.

In the present context, the term “fluid flow of retentate” relates to the flow of the retentate when entering the tangential flow filtration unit. Thus, if the retentate is not re-circulated or not fully re-circulated, this fluid flow would relate to the fluid flow of the mixture or biological composition when entering the tangential flow filtration unit.

In the present context, the term “permeate” relates to a composition which has passed through the pores of a filter. Thus, a permeate is the part of a filtered composition which went through the filter i.e. passed through the filter to the permeate side of the filter.

In the present context, the term “fluid flow of permeate” relates to the flow of permeate measured either in the third fluid connection or at the first outlet.

In the present context, the term “continuous tangential fluid flow” relates to having a tangential flow throughout all the process steps.

In the present context, the term “backward flush”, or “back flush”, relates to an event wherein the flow of composition through the pores of the filter is reversed i.e. from the permeate site of the membrane to the retentate site.

Pulsed Pressure Increase and/or Backward Flush of the Permeate

In the present context, the term “pulsed pressure increase and/or backward flush of the permeate” relates to an event wherein the pressure on the permeate side is increased. For example, the pressure increase may be large enough that the difference between the pressure on the retentate side and the pressure on the permeate side of the filter is essentially zero, whereby the flow of composition through the filter may be reversed to give a weak backward flush though the filter. In another example, the pressure increase on the permeate side is much larger than the pressure on the retentate side, whereby a strong backward flush is applied.

In the present context, the term “biological composition” refers to a complex composition obtained from a biological source, such as plants, microorganisms and animals, and being a liquid, suspension or blend. For example, the biological composition may be milk, whey, fermentation broth, plant extracts or blood plasma.

In the present context, the term “target compound” refers to the compound in the biological composition, which is to be separated from the biological composition.

The target compound may be any compound of interest such as coloured molecules, nucleotides, polyphenols, metal ions, toxic compounds and biomolecules such as proteins and peptides.

In a preferred embodiment, the target compound is a target protein.

In the present context, the term “mixture” refers to a biological composition comprising a target compound which is mixed with sorption material.

In the present context, the term “refining” refers to a process wherein a compound is concentrated, separated, cleaned, purified, and/or isolated from a biological composition.

In the present context, the term “separated” refers to something which has been set apart from something else. More specifically, it may be a compound which has been set apart from other species of a composition, such that a new composition comprising the compound is obtained and another new composition not comprising the compound is obtained. In some applications the new composition not comprising the species bound to the adsorbent may (also) be a/the target product.

In the present context, the term “transmembrane pressure” is defined as:

The present invention provides an apparatus for refining a compound comprised in a biological composition.

By reference tobut without being limited hereby, a first aspect of the present invention relates to an apparatus comprising a first inlet (); a tangential flow filtration unit () comprising a membrane () having a retentate site () and a permeate site () and a nominal pore size in the range of 1 to 1000 μm; one or more first fluid connections () leading fluid from said first inlet () to said tangential flow filtration unit (); one or more second fluid connections () leading retentate from said tangential flow filtration unit () to said first inlet () and/or said first fluid connections (); a circulation pump () for re-circulation of fluid through said tangential flow filtration unit () via said first and second fluid connections (,); one or more third fluid connections () for leading permeate to a first outlet (); and a sorption material, wherein said sorption material comprising chemically modified porous particles having an average particle size of at least 5 micron, such as at least 10 micron, such as at least 20 micron; and said apparatus, when in use, comprises a dynamic layer of said sorption material on the retentate side () of said membrane (), said dynamic sorption material layer being capable of reaching a thickness of at least 1 mm, such as at least 3 mm, such as at least 5 mm, such as at least 10 mm, such as at least 15 mm, such as at least 20 mm, such as at least 25 mm, such as at least 30 mm, such as at least 35 mm, such as at least 40 mm.

shows the circulation pump () being arranged in the first fluid connection (). However, the circulation pump () may as well be arranged in the second fluid connection () as long as the circulation pump () is adapted for re-circulating the retentate in the apparatus. Thus, in one embodiment, the circulation pump () is arranged in the first fluid connection (). In a further embodiment, the circulation pump () is arranged in the second fluid connection ().

The apparatus forms a dynamic layer of the sorption material on the retentate side of the membrane once the process is initiated. The layer is gradually formed during the process. Forming a layer of sorption material results in a more efficient separation process as well as a process demanding less resources for washing and elution.illustrates the formation of a dynamic layer of sorption material on the retentate side () of the membrane (). The dynamic layer is formed by sorption particles () reaching a given thickness ().

Alternatively, said dynamic layer could be present in the apparatus prior to use having been formed during a previous run.

The tangential flow filtration unit () may be equipped with any type of membrane (). The third fluid connection () leading permeate to outlet is connected to the first outlet () that may direct the permeate for collection in a waste tank or in a permeate collection tank. It is to be understood that in one embodiment, the outlet is to be understood merely as an opening from which permeate may leave the apparatus, e.g. as shown in.

shows a more detailed schematic representation of an apparatus according to an embodiment of the present invention. In this embodiment, a permeate pump () is inserted in the third fluid connection () for controlling the fluid flow of the permeate. Hence, in one embodiment, the apparatus further comprises a permeate pump (). In a further embodiment, the permeate pump () is arranged in connection with the one or more third fluid connections ().